Genomic instability is one of the hallmarks of cancer cells. We are studying telomere function to both understand how chromosomes are maintained and to explore their role in the initiation and growth of cancer cells. Telomeres distinguish natural chromosome ends from double stranded DNA breaks. They maintain chromosome stability by protecting chromosome ends from processes that normally occur at breaks such as fusion, translocation and DNA degradation. Telomeres are maintained by telomerase, which adds the simple sequence repeat TTAGGG onto all ends. In the next five years we will take advantage of the telomerase null mouse, mTR-/-, to dissect the role of telomeres in tumor initiation and growth. Using this mouse we previously established that under some conditions short telomeres induce apoptosis and thus decrease tumor growth. In other genetic settings, however, short telomeres initiate chromosomal rearrangements and increase tumor formation. We will examine the proteins that influence which pathway will predominate in cells that have short, dysfunctional telomeres. We will use shRNAs to knock down the level of specific proteins that signal DNA damage, to determine their role in signaling the loss of telomere function. We will examine the role of these genes in both cultured cells in vitro and in tumor formation. To identify new proteins that may recognize dysfunctional telomeres we will screen for shRNAs that interfere with the response to short telomeres. Although telomerase is required for the growth of many tumor cells, some cells have mechanisms that maintain telomeres (alternative telomere lengthening , ALT) and allow growth in the absence of telomerase. We will examine which recombination pathway(s) play a role in the growth of ALT cells and whether interfering with these pathways inhibits the growth of mTR-/- tumors. Understanding the role of these ALT pathways in tumors is essential for potential cancer therapeutics since these alternative mechanisms will be selected for in human tumors that are treated with telomerase inhibitors. Finally, we will examine the role of telomerase activity in stem cells and how that may potentiate stem cell derived tumors. The hedgehog signaling pathway, which is active in many stem cells, is required for the growth of a variety of common human tumors. We will examine whether telomerase is a target of the hedgehog signaling pathway and what role telomerase activity plays in these stem cell derived tumors.